LOCUS       sequence-355619-        6194 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 6194)
  AUTHORS   Jin J, Li X, Gygi SP, Harper JW
  TITLE     Dual E1 activation systems for ubiquitin differentially regulate E2 
            enzyme charging.
  JOURNAL   Nature. 2007 Jun 28. 447(7148):1135-8.
  PUBMED    17597759
REFERENCE   2  (bases 1 to 6194)
  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: "Nature. 
            2007 Jun 28. 447(7148):1135-8."
COMMENT     Sequence Label: pET15-UbE2W
FEATURES             Location/Qualifiers
     source          1..6194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(696..714)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      710..757
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(932..937,938..954,955..960))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(987..1005)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1073..1177
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1178..1246,1247..2038)
                     /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     complement(1396..1415)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2209..2797
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2698..2717
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2951..2968
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2983..3122
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3048..3067)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3208..3230
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3224..3415)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5770..5789
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5809..5886)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6092..6111
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg ctcgagatgg cgtcaatgca gaccacagga
      181 aggagggtgg aagtatggtt tccaaaacga ctacagaaag aactgttggc tttgcaaaat
      241 gacccacctc ctggaatgac cttaaatgag aagagtgttc aaaattcaat tacacagtgg
      301 attgtagaca tggaaggtgc accaggtacc ttatatgaag gggaaaaatt tcaacttcta
      361 tttaaattta gtagtcgata tccttttgac tctcctcagg tcatgtttac tggtgaaaat
      421 attcctgttc atcctcatgt ttatagcaat ggtcatatct gtttatccat tctaacagaa
      481 gactggtccc cagcgctctc agtccaatca gtttgtctta gcattattag catgctttcc
      541 agctgcaagg aaaagagacg accaccggat aattcttttt atgtgcgaac atgtaacaag
      601 aatccaaaga aaacaaaatg gtggtatcat gatgatactt gttgaggatc cggctgctaa
      661 caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc
      721 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg
      781 atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac agcatccagg
      841 gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg tgcctgactg
      901 cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat aagctgtcaa
      961 acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     1021 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     1081 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1141 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     1201 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     1261 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     1321 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     1381 cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca
     1441 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     1501 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     1561 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     1621 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     1681 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt
     1741 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     1801 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     1861 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     1921 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     1981 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     2041 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     2101 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     2161 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     2221 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     2281 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     2341 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     2401 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     2461 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     2521 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     2581 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     2641 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     2701 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     2761 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     2821 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     2881 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     2941 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     3001 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     3061 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     3121 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     3181 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     3241 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     3301 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     3361 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     3421 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     3481 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     3541 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     3601 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     3661 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     3721 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     3781 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     3841 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac ccaacgctgc
     3901 ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg ttctgccaag
     3961 ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt cttggagtgg
     4021 tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc
     4081 accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa tccatgccaa
     4141 cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg gtccaatgat
     4201 cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat ggtcgtcatc
     4261 tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg aagcgagaag
     4321 aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga cgtagcccag
     4381 cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4441 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4501 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4561 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4621 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4681 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4741 ccagtcggga aactgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4861 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4921 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4981 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5041 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5101 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5161 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5221 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5281 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5341 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5401 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5461 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5521 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5581 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5641 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5701 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5761 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5821 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5881 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5941 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6001 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6061 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6121 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     6181 cgatcccgcg aaat
//
