LOCUS       sequence-218961-        6455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of HUWE1 HECT domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6455)
  AUTHORS   Michel MA, Swatek KN, Hospenthal MK, Komander D
  TITLE     Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked 
            Ubiquitin Signaling.
  JOURNAL   Mol Cell. 2017 Oct 5;68(1):233-246.e5. doi: 
            10.1016/j.molcel.2017.08.020. Epub 2017 Sep 21.
  PUBMED    28943312
REFERENCE   2  (bases 1 to 6455)
  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: 
            "10.1016/j.molcel.2017.08.020"; journalName: "Mol Cell"; date: 
            "2017-10-5- 5"; volume: "68"; issue: "1"; pages: "233-246.e5"
COMMENT     Sequence Label: Human HUWE1 (3993-4374)
FEATURES             Location/Qualifiers
     source          1..6455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             308..325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             335..358
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1528..1545
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1598..1616)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1612..1659
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1696..2151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1783..1802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1993..2014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2243..3058)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2243..3058)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2966..2985
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3050..3069
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3180..3768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3669..3688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3922..3939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3954..4093
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4019..4038)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4179..4201
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4195..4386)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5195..6277)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6239..6258
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6278..6355)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat ataccatggg
      301 cagcagccat catcatcatc atcacagcag cggtctggaa gttctgtttc agggcccgga
      361 ctttgatgtc aagcgcaaat atttccgcca agagctggag cgtttagatg aggggctccg
      421 gaaagaagac atggctgtgc atgtccgtcg tgaccatgtg tttgaagact cctatcgtga
      481 gctgcatcgc aaatcccccg aagaaatgaa gaatcgattg tatatagtat ttgaaggaga
      541 agaagggcag gatgctggcg ggctcctgcg ggagtggtat atgatcatct ctcgagagat
      601 gtttaaccct atgtatgcct tgttccgtac ctcacctggt gatcgagtca cctacaccat
      661 caatccatct tcccactgca accccaacca cctcagctac ttcaagtttg tcggacgcat
      721 tgtggccaaa gctgtatatg acaaccgtct tctggagtgc tactttactc gatcctttta
      781 caaacacatc ttgggcaagt cagtcagata tacagatatg gagagtgaag attaccactt
      841 ctaccaaggt ctggtttatc tgctggaaaa tgatgtctcc acactaggct atgacctcac
      901 cttcagcact gaggtccaag agtttggagt ttgtgaagtt cgtgacctca aacccaatgg
      961 ggccaacatc ttggtaacag aggagaataa gaaggagtat gtacacctgg tatgccagat
     1021 gagaatgaca ggagccatcc gcaagcagtt ggcggctttc ttagaaggct tctatgagat
     1081 cattccaaag cgcctcattt ccatcttcac tgagcaggag ttagagctgc ttatatcagg
     1141 actgcccacc attgacatcg atgatctgaa atccaacact gaataccaca agtaccagtc
     1201 caactctatt cagatccagt ggttctggag agcattgcgt tctttcgatc aagctgaccg
     1261 tgccaagttc ctccagtttg tcacgggtac ttccaaggta cccctgcaag gctttgctgc
     1321 cctcgaaggc atgaatggca ttcagaagtt tcagatccat cgagatgaca ggtccacaga
     1381 tcgcctgcct tcagctcaca catgttttaa tcagctggat ctgcctgcct atgagagctt
     1441 tgagaagctc cgccacatgc tactgttggc tatccaggag tgctctgaag gctttgggct
     1501 ggcctaaagc tttctagacc atttaaacac caccaccacc accactgaga tccggctgct
     1561 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1621 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1681 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     1741 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     1801 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1861 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1921 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1981 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2041 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2101 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2161 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2221 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     2281 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     2341 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     2401 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     2461 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     2521 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     2581 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     2641 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     2701 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     2761 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     2821 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2881 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2941 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3001 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3061 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3121 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3301 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     3361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3781 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3841 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3901 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3961 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4021 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4081 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4141 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4201 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4261 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     4321 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     4381 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     4441 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     4501 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     4561 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     4621 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     4681 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     4741 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     4801 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4861 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4921 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4981 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5041 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5101 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5161 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5221 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5281 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     5341 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     5401 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     5461 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     5521 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     5581 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     5641 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     5701 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     5761 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     5821 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5881 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5941 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6001 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6061 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6121 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6181 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     6241 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     6301 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     6361 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     6421 ccgttgagca ccgccgccgc aaggaatggt gcatg
//
