LOCUS       sequence-441365-        5313 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 6xHIS-tagged Ubiquitin containing K48R mutation in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5313)
  AUTHORS   Jones RM, Ruiz JH, Scaramuzza S, Nath S, Liu C, Henklewska M, 
            Natsume T, Bristow RG, Romero F, Kanemaki MT, Gambus A
  TITLE     Characterizing replisome disassembly in human cells.
  JOURNAL   iScience. 2024 Jun 12;27(7):110260. doi: 10.1016/j.isci.2024.110260.
            eCollection 2024 Jul 19.
  PUBMED    39055910
REFERENCE   2  (bases 1 to 5313)
  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: "iScience.";
            date: "2024-06-12"; volume: "27(7):110260. doi"; pages: " 
            10.1016/j.isci.2024.110260. eCollection 2024 Jul 19"
COMMENT     Sequence Label: pcDNA5 HIS-Ub-K48R
FEATURES             Location/Qualifiers
     source          1..5313
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(507..1298,1299..1367))
                     /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     1130..1149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1368..1472)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1547..1566)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1738..2117
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2118..2321
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2272..2292
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2323..2341
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2344..2362
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2372..2396
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2482..2499
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2500..2727
                     /codon_start=1
                     /product="human ubiquitin variant in which Lys-48 has been 
                     mutated to arginine"
                     /label=ubiquitin-K48R
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     primer_bind     complement(2768..2785)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2774..2998
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3131..3150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3282..3329
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3337..4359
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3337..4359
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4489..4610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4526..4545)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4580..4599
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4659..4675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4659..4675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4672..4694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4683..4699
                     /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(4707..4713,4714..4731,4732..4737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4752..4773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4890..4907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5061..5313,1..336))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5141..5160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
       61 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      121 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      181 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      241 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      301 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      361 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      421 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      481 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      541 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
      601 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
      661 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
      721 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
      781 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
      841 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
      901 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
      961 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1021 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1081 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1141 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1201 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1261 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1321 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1381 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     1441 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     1501 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     1561 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     1621 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     1681 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
     1741 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     1801 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     1861 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     1921 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     1981 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2041 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2101 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     2161 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     2221 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     2281 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctccctatc agtgatagag
     2341 atctccctat cagtgataga gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct
     2401 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc
     2461 ggactctagc gtttaaacat gcatcaccat caccatcaca tgcagatctt cgtgaagacc
     2521 ctgactggta agaccatcac tctcgaagtg gagccgagtg atacgatcga aaacgtcaag
     2581 gcaaagatcc aagacaagga aggcatccct cctgaccagc agaggttgat ctttgctggg
     2641 agacagctgg aagatggacg caccctgtct gactacaaca tccagaaaga gtccaccctg
     2701 cacctggtcc tccgtctcag aggtgggtag ctcgagtcta gagggcccgt ttaaacccgc
     2761 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2821 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2881 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2941 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3001 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3061 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3121 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3181 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3241 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     3301 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     3361 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     3421 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     3481 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     3541 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     3601 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     3661 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     3721 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     3781 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     3841 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     3901 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     3961 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     4021 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     4081 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     4141 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     4201 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     4261 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     4321 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     4381 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4441 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4501 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4561 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4621 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4681 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4741 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4801 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4861 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4921 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4981 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5041 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5101 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5161 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5221 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5281 tcggtgtagg tcgttcgctc caagctgggc tgt
//
