LOCUS       sequence-233021-        8828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human Rad18 and BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8828)
  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 8828)
  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-Rad18
FEATURES             Location/Qualifiers
     source          1..8828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        140..388
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     140..160
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     311..330
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        410..485
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        520..899
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          1179..2195
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2119..2141
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2208..2227
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2208..2226
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2228..2237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3739..3755)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3775..3999
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     intron          4220..5158
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5115..5135
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      5193..5202
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5199..5201,5202..5204,5205..5918)
                     /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(5244..5265)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    5949..6060
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6178..6195)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6178..6194)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6187..6209)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(6335..6790)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6472..6493)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6684..6703
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6817..6921
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6922..6990,6991..7782)
                     /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(7140..7159)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7953..8541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8442..8461
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8695..8712
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ggcgcgcccc cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg
      181 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      241 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      301 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      361 atatatcttg tggaaaggac gaaacaccga ctccagtctt tctagaagag ttttagagct
      421 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      481 ggtgcttttt ttgatccaag ggtggggcgc gccggattcg acattgatta ttgactagtt
      541 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
      601 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
      661 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
      721 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
      781 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
      841 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
      901 tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa
      961 ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg
     1021 ggcgcgcgcc rgggggggsg gggggggggg gggggggggg sggggsgrgg sggrgrggkg
     1081 sggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc
     1141 ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc
     1201 gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt
     1261 tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg
     1321 tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg
     1381 gccctttgtg cggggggagc ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg
     1441 ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt
     1501 gtgcgctccg cagtgtgcgc gaggggagcg cggccggggg cggtgccccg cggtgcgggg
     1561 ggggctgcga ggggaacaaa ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg
     1621 gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc ccctccccga gttgctgagc
     1681 acggcccggc ttcgggtgcg gggctccgta cggggcgtgg cgcggggctc gccgtgccgg
     1741 gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggrgg
     1801 gctcgggggr ggggcgcggc ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc
     1861 gcagccattg ccttttatgg taatcgtgcg agagggcgca gggacttcct ttgtcccaaa
     1921 tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcggggcga
     1981 agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc
     2041 gccgtcccct tctccctctc cagcctcggg gctgtccgcg gggggacggc tgccttcggg
     2101 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     2161 ctaaccatgt tcatgccttc ttctttttcc tacagatcct taattaataa tacgactcac
     2221 tataggggcc accatggact ccctggccga gtctcggtgg cctccgggcc tggcagtcat
     2281 gaagacaata gatgatttgc tgcggtgtgg aatttgcttc gagtatttca acattgcaat
     2341 gataatacct cagtgttcac ataactactg ctctctctgt ataagaaaat ttctgtccta
     2401 taaaactcag tgtccaactt gctgtgtgac tgtcacagag ccggatctga aaaataaccg
     2461 catattagat gaactggtaa aaagcttgaa ttttgcacgg aatcatctgc tgcagtttgc
     2521 tttagagtca ccagccaaat ctcctgcttc ttcctcttca aagaatcttg ctgtcaaagt
     2581 atatactcct gtagcctcca gacagtcttt aaagcagggg agcaggttaa tggataattt
     2641 cttgatcaga gaaatgagtg gttctacatc agagttgttg ataaaagaaa ataaaagcaa
     2701 attcagccct caaaaagagg cgagccctgc tgcaaagacc aaagagacac gttctgtaga
     2761 agagatcgct ccagatccct cagaggctaa gcgtcctgag ccaccctcga catccacttt
     2821 gaaacaagtt actaaagtgg attgtcctgt ttgcggggtt aacattccag aaagtcacat
     2881 taataagcat ttagacagct gtttatcacg cgaagagaag aaggaaagcc tcagaagttc
     2941 tgttcacaaa aggaagccgc tgcccaaaac tgtatataat ttgctctctg atcgtgattt
     3001 aaagaaaaag ctaaaagagc atggattatc tattcaagga aataaacaac agctcattaa
     3061 aaggcaccaa gaatttgtac acatgtacaa tgcccaatgc gatgctttgc atcctaaatc
     3121 agctgctgaa atagttcaag aaatcgaaaa tatagagaag actaggatgc gtcttgaagc
     3181 tagtaaactc aatgaaagtg taatggtttt tacaaaggac caaacagaaa aggaaataga
     3241 tgaaatccac agtaaatatc gtaaaaaaca taagagtgaa tttcagcttc tggtggatca
     3301 ggctagaaaa ggatacaaga aaattgctgg aatgtcacaa aaaacagtaa caataacaaa
     3361 agaagatgaa tctacagaaa agctatcttc tgtatgcatg ggacaggaag ataatatgac
     3421 ctcagtaaca aaccactttt ctcaatcaaa gctggactcc ccagaggaat tggaacctga
     3481 cagagaagag gattcttcta gctgtattga tattcaagaa gttctttctt catcagaatc
     3541 agattcatgc aatagttcca gttcagacat cataagagat cttttagaag aagaggaagc
     3601 ctgggaagca tcacataaaa acgatcttca agacacagaa ataagtccaa gacagaatcg
     3661 ccgcacaaga gccgctgaaa gtgctgagat tgaaccaaga aacaagcgta ataggaatta
     3721 aacgcgtaaa tgattgcaga tccactagtt ctagagctcg ctgatcagcc tcgactgtgc
     3781 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3841 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3901 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3961 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     4021 gctggggctc gagatccact agttctagcc tcgaggctag agcggccccg gtgcctagag
     4081 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     4141 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     4201 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     4261 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga
     4321 tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc
     4381 ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg
     4441 gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg
     4501 atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct
     4561 gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg
     4621 cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc
     4681 tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg
     4741 ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg
     4801 ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc
     4861 acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga
     4921 gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt
     4981 aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga
     5041 agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg
     5101 atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt
     5161 gtcgtgagcg atcgcatcga cagtgtttaa acgccaccat ggtgagcaag ggcgaggagc
     5221 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     5281 tcagcgtgag gggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca
     5341 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgagccacg
     5401 gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     5461 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcacctaca
     5521 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     5581 gcgtcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca
     5641 gccacaacat ctatatcatg gccgtcaagc agaagaacgg catcaaggtg aacttcaaga
     5701 tccgccacaa cgtggaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     5761 ccatcggcga cggccccgtg ctgctgcccg acagccacta cctgagcacc cagtccgtgc
     5821 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc cgcaccgccg
     5881 ccgggatcac tctcggcatg gacgagctgt acaagtgaga attcctagag ctcgctgatc
     5941 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     6001 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     6061 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     6121 ggaggattgg gaagagaata gcaggcatgc gtttaaacat tagtcgacag ggccgccact
     6181 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     6241 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     6301 ttcccaacag ttgcgcagcc tgaatggcga atggaaattg taagcgttaa tattttgtta
     6361 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     6421 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     6481 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     6541 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     6601 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6661 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6721 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6781 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6841 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6901 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6961 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     7021 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     7081 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     7141 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     7201 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     7261 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     7321 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     7381 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     7441 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     7501 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     7561 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7621 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     7681 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7741 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7801 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7861 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7921 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7981 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     8041 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     8101 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     8161 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     8221 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     8281 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     8341 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     8401 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     8461 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     8521 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8581 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     8641 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     8701 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     8761 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     8821 aacgcaat
//
