LOCUS       sequence-176967-        5956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5956)
  AUTHORS   Brzovic PS, Lissounov A, Christensen DE, Hoyt DW, Klevit RE
  TITLE     A UbcH5/ubiquitin noncovalent complex is required for processive 
            BRCA1-directed ubiquitination.
  JOURNAL   Mol Cell. 2006 Mar 17. 21(6):873-80.
  PUBMED    16543155
REFERENCE   2  (bases 1 to 5956)
  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: "Mol Cell. 
            2006 Mar 17. 21(6):873-80."
COMMENT     Sequence Label: Ubiquitin WT
FEATURES             Location/Qualifiers
     source          1..5956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(346..363)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(373..390)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(417..644)
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     RBS             651..673
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    688..712
                     /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     complement(712..731)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(713..731)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(815..834)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1040..1117
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1137..1156)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3511..3702
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3696..3718)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3804..3943
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3859..3878
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3958..3975)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4129..4717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4209..4228)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4888..5679,5680..5748))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5511..5530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5749..5853)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5921..5939
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atcgtcgacg atcctcgagc atatggctgc cgcgcggcac
      361 caggccgctg ctgtgatgat gatgatgatg gctgctgccc atggggtacc tcatcaacca
      421 cctcttagtc ttaagacaag atgtaaggtc gactccttct gaatgttgta atcagacagc
      481 gttctaccgt cctcgagctg cttaccggca aagatcaatc tttgttgatc aggtggaatt
      541 ccttccttgt cttgaatttt agccttaacg ttttcgatgg tatcggatgg ttcaacttcc
      601 agagttatgg ttttaccggt taacgtcttg acgaagatct gcatggatcc tctccttctt
      661 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
      721 gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc
      781 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
      841 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
      901 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg
      961 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     1021 ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg
     1081 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     1141 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     1201 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     1261 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     1321 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     1381 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     1441 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     1501 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     1561 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     1621 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     1681 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     1741 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     1801 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     1861 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
     1921 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     1981 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     2041 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     2101 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     2161 gctggcacga cagtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     2221 ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt
     2281 cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
     2341 tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg
     2401 ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc
     2461 cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat
     2521 tatcgccggc atggcggccg acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg
     2581 ctggatggcc ttccccatta tgattcttct cgcttccggc ggcatcggga tgcccgcgtt
     2641 gcaggccatg ctgtccaggc aggtagatga cgaccatcag ggacagcttc aaggatcgct
     2701 cgcggctctt accagcctaa cttcgatcat tggaccgctg atcgtcacgg cgatttatgc
     2761 cgcctcggcg agcacatgga acgggttggc atggattgta ggcgccgccc tataccttgt
     2821 ctgcctcccc gcgttgcgtc gcggtgcatg gagccgggcc acctcgacct gaatggaagc
     2881 cggcggcacc tcgctaacgg attcaccact ccaagaattg gagccaatca attcttgcgg
     2941 agaactgtga atgcgcaaac caacccttgg cagaacatat ccatcgcgtc cgccatctcc
     3001 agcagccgca cgcggcgcat ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc
     3061 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
     3121 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
     3181 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
     3241 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
     3301 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
     3361 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
     3421 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
     3481 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
     3541 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
     3601 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
     3661 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     3721 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     3781 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     3841 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cattgcggtg
     3901 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc
     3961 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4021 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4081 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4141 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4201 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4261 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4321 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4381 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4441 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4501 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4561 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4621 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4681 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4741 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4801 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4861 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4921 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4981 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5041 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5101 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5161 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc
     5221 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5281 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5341 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5401 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5461 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc
     5521 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5581 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5641 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5701 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5821 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5881 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5941 ctttcgtctt caagaa
//
