LOCUS       sequence-258776-        4832 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 4832)
  AUTHORS   Raasi S, Pickart CM
  TITLE     Ubiquitin chain synthesis.
  JOURNAL   Methods Mol Biol. 2005;301:47-55. doi: 10.1385/1-59259-895-1:047.
  PUBMED    15917625
REFERENCE   2  (bases 1 to 4832)
  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: "Methods Mol
            Biol."; date: "2005"; volume: "301:47-55. doi: 
            10.1385/1-59259-895-1"; pages: "047"
COMMENT     Sequence Label: pET3a-Ub-K63R
FEATURES             Location/Qualifiers
     source          1..4832
                     /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(404..451)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     447..465
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(519..746)
                     /codon_start=1
                     /product="human ubiquitin variant in which Lys-63 has been 
                     mutated to arginine"
                     /label=ubiquitin-K63R
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQRESTLHLVLRLRGG"
     CDS             complement(519..746)
                     /codon_start=1
                     /product="human ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQRESTLHLVLRLRGG"
     RBS             754..776
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(807..826)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(808..826)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(910..929)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             2387..2578
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2572..2594)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2680..2819
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2735..2754
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2834..2851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3005..3593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3085..3104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3764..4555,4556..4624))
                     /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     4387..4406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4625..4729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4797..4815
                     /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 gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata
      241 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg
      301 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac
      361 cacacccgtc ctgtggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
      421 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
      481 cagcttcctt tcgggctttg ttagcagccg gatcctcaac cacctcttag tcttaagaca
      541 agatgtaagg tcgactcccg ctgaatgttg taatcagaca gcgttctacc gtcctcgagc
      601 tgcttaccgg caaagatcaa tctttgttga tcaggtggaa tgccttcctt gtcttgaatt
      661 ttagccttaa cgttttcgat ggtatcggat ggttcaactt ctagagttat ggttttaccg
      721 gttaacgtct tgacgaagat ctgcatatgt atatctcctt cttaaagtta aacaaaatta
      781 tttctagagg gaaaccgttg tggtctccct atagtgagtc gtattaattt cgcgggatcg
      841 agatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
      901 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
      961 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     1021 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     1081 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgaccg atgcccttga
     1141 gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac
     1201 ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca
     1261 ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat
     1321 tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg
     1381 gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac gtcttgctgg
     1441 cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca
     1501 tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac
     1561 agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcattgga ccgctgatcg
     1621 tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg attgtaggcg
     1681 ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct
     1741 cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa gaattggagc
     1801 caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga acatatccat
     1861 cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg ggtcctggcc
     1921 acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
     1981 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
     2041 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
     2101 ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
     2161 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt
     2221 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag
     2281 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg
     2341 tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga
     2401 aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa
     2461 actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc
     2521 tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2581 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2641 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     2701 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     2761 gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     2821 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     2881 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     2941 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     3001 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     3061 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     3121 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     3181 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     3241 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     3301 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     3361 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     3421 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     3481 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     3541 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     3601 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     3661 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     3721 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     3781 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     3841 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     3901 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     3961 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     4021 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg
     4081 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     4141 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     4201 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     4261 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     4321 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     4381 cacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     4441 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     4501 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     4561 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     4621 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     4681 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     4741 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     4801 ggcgtatcac gaggcccttt cgtcttcaag aa
//
