LOCUS       sequence-224973-        7121 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the human RNF169 Ubiquitin binding domain 
            and BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7121)
  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 7121)
  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..7121
                     /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"
     promoter        419..696
                     /label=chicken beta-actin promoter
     intron          697..1708
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1632..1654
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1721..1740
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1721..1739
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1741..1750
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1750..1773
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1935..1951)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1971..2195
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          2416..3354
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3311..3331
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      3389..3398
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3395..3397,3398..3400,3401..4114)
                     /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(3440..3461)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    4145..4256
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4374..4391)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4374..4390)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4383..4405)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4532..4987
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4619..4638)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4829..4850
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5013..5117
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5118..5186,5187..5978)
                     /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(5336..5355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6149..6737
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6638..6657
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6891..6908
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7025..7046
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7061..7066,7067..7084,7085..7091)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7099..7115
                     /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(7104..7121,1..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 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg
      601 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      661 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgtc gcgccttcgc
      721 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      781 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      841 taatgacggc tcgtttcttt tctgtggctg cgtgaaagcc ttaaagggct ccgggagggc
      901 cctttgtgcg ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
      961 cgcgtgcggc ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
     1021 gcgctccgcg tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg
     1081 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     1141 gggcgcggcg gtcgggctgt aacccccccc tgcacccccc tccccgagtt gctgagcacg
     1201 gcccggcttc gggtgcgggg ctccgtgcgg ggcgtggcgc ggggctcgcc gtgccgggcg
     1261 gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc ggggagggct
     1321 cgggggaggg gcgcggcggc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     1381 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     1441 gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg gcgaagcggt
     1501 gcggcgccgg caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc
     1561 cccttctcca tctccagcct cggggctgcc gcagggggac ggctgccttc gggggggacg
     1621 gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct ctgctaacca
     1681 tgttcatgcc ttcttctttt tcctacagat ccttaattaa taatacgact cactataggg
     1741 gccaccatgg actacaagga tgacgacgat aaaaagcttc agcaagagga agaagaccga
     1801 cagttggctc tgcagttgca gcgcatgttc gacaatgaga ggcggactgt gagccggcga
     1861 aaaggaagtg tggatcagta tctcctacgg tccagcaaca tggccggggc caagtagacg
     1921 cgtaaatgat tgcagatcca ctagttctag agctcgctga tcagcctcga ctgtgccttc
     1981 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     2041 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     2101 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     2161 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     2221 gggctcgaga tccactagtt ctagcctcga ggctagagcg gccccggtgc ctagagaagg
     2281 tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt
     2341 gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt
     2401 gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
     2461 tatggccctt gcgtgccttg aattacttcc acctggctgc agtacgtgat tcttgatccc
     2521 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
     2581 cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg
     2641 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
     2701 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
     2761 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
     2821 tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
     2881 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
     2941 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
     3001 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     3061 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     3121 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     3181 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     3241 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     3301 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     3361 tgagcgatcg catcgacagt gtttaaacgc caccatggtg agcaagggcg aggagctgtt
     3421 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     3481 cgtgaggggc gagggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg
     3541 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga gccacggcgt
     3601 gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     3661 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca cctacaagac
     3721 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcgt
     3781 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact tcaacagcca
     3841 caacatctat atcatggccg tcaagcagaa gaacggcatc aaggtgaact tcaagatccg
     3901 ccacaacgtg gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     3961 cggcgacggc cccgtgctgc tgcccgacag ccactacctg agcacccagt ccgtgctgag
     4021 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttccgca ccgccgccgg
     4081 gatcactctc ggcatggacg agctgtacaa gtgagaattc ctagagctcg ctgatcagcc
     4141 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4201 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4261 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4321 gattgggaag agaatagcag gcatgcgttt aaacattagt cgacagggcc gccactggcc
     4381 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     4441 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     4501 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     4561 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     4621 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4681 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4741 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4801 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4861 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     4921 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     4981 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5041 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5101 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5161 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5221 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     5281 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     5341 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     5401 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5461 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5521 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5581 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5641 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5701 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5761 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5821 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5881 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5941 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6001 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6061 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6121 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6181 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6241 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     6301 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6361 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6421 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6481 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6541 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6601 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6661 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6721 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6781 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6841 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6901 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     6961 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     7021 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     7081 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca c
//
