LOCUS       sequence-151348-        5674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Ubiquitin variant with mutations L69P and V70L reverted to wild type
            (i53-DM mutant).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5674)
  AUTHORS   Canny MD, Moatti N, Wan LCK, Fradet-Turcotte A, Krasner D, 
            Mateos-Gomez PA, Zimmermann M, Orthwein A, Juang YC, Zhang W, 
            Noordermeer SM, Seclen E, Wilson MD, Vorobyov A, Munro M, Ernst A, 
            Ng TF, Cho T, Cannon PM, Sidhu SS, Sicheri F, Durocher D
  TITLE     Inhibition of 53BP1 favors homology-dependent DNA repair and 
            increases CRISPR-Cas9 genome-editing efficiency.
  JOURNAL   Nat Biotechnol. 2018 Jan;36(1):95-102. doi: 10.1038/nbt.4021. Epub 
            2017 Nov 27.
  PUBMED    29176614
REFERENCE   2  (bases 1 to 5674)
  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"; doi: "10.1038/nbt.4021"; 
            journalName: "Nat Biotechnol"; date: "2018-01"; volume: "36"; issue:
            "1"; pages: "95-102"
COMMENT     Sequence Label: pcDNA3-Flag::UbvG08 P69L, L70V, I44A, deltaGG
FEATURES             Location/Qualifiers
     source          1..5674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             904..927
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(1226..1244)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1227..1244)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1264..1281)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1270..1494
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1540..1968
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1627..1646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1837..1858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1977..1997)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1982..2312
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2163..2298
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2225..2244
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2379..3173
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2433..2452)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3043..3062
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3347..3468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3384..3403)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3438..3457
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3517..3533)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3517..3533)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3530..3552)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3541..3557
                     /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)."
     promoter        complement(join(3565..3571,3572..3589,3590..3595))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3610..3631
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3748..3765)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3919..4507)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3999..4018)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4678..5469,5470..5538))
                     /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     5301..5320
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5539..5643)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 atggactaca aagacgatga cgataaagcc gccagtttaa acggcgcgcc attaattaag
      961 gatccaatgt tgattttcgt gaaaaccctt accgggaaaa ccatcaccct cgaggttgaa
     1021 ccctcggata cgatagaaaa tgtaaaggcc aagatccagg ataaggaagg aattcctcct
     1081 gatcagcaga gactggcctt tgctggcaaa tcgctggaag atggacgtac tttgtctgac
     1141 tacaatattc taaaggactc taaacttcat ctagtgttga gacttcgttg agcggccgct
     1201 cgagcatgca tctagagggc cctattctat agtgtcacct aaatgctaga gctcgctgat
     1261 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     1321 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     1381 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     1441 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     1501 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     1561 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1621 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1681 aagctctaaa tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1741 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1801 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1861 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg
     1921 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     1981 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca ggcaggcaga agtatgcaaa
     2041 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2101 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2161 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     2221 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     2281 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     2341 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     2401 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     2461 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     2521 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     2581 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     2641 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     2701 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     2761 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     2821 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     2881 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     2941 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3001 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3061 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3121 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3181 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     3241 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     3301 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     3361 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     3421 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     3481 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3541 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3601 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3661 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3721 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3781 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3841 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3901 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3961 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4021 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4081 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     4141 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4201 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4261 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4321 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     4381 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4441 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4501 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4561 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4621 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4681 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4741 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4801 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4861 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4921 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4981 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5041 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5101 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5161 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5221 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5281 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5341 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5401 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5461 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5521 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5581 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5641 gcgcacattt ccccgaaaag tgccacctga cgtc
//
