LOCUS       sequence-369325-        7918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  insect cell expression of HUWE1 HECT domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7918)
  AUTHORS   Hunkeler M, Jin CY, Ma MW, Monda JK, Overwijn D, Bennett EJ, Fischer
            ES
  TITLE     Solenoid architecture of HUWE1 contributes to ligase activity and 
            substrate recognition.
  JOURNAL   Mol Cell. 2021 Sep 2;81(17):3468-3480.e7. doi: 
            10.1016/j.molcel.2021.06.032. Epub 2021 Jul 26.
  PUBMED    34314700
REFERENCE   2  (bases 1 to 7918)
  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.2021.06.032"; journalName: "Mol Cell"; date: 
            "2021-09-2- 2"; volume: "81"; issue: "17"; pages: "3468-3480.e7"
COMMENT     Sequence Label: pAC8-HUWE1-HECTonly
FEATURES             Location/Qualifiers
     source          1..7918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1342..1797
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1429..1448)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1639..1660
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1814..1833)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1933..1955
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1993..2011)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2079..2183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2184..2252,2253..3044)
                     /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     complement(2402..2421)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3215..3803
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3704..3723
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3957..3974
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_recomb     4366..5193
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             4366..4490
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(4528..5133)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     protein_bind    complement(5197..5230)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    5420..5468
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     5493..5512
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(5546..6223)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Strongin et al., 2007)"
                     /label=DsRed-Monomer
                     /note="mammalian codon-optimized"
                     /translation="MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGV
                     VEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISH
                     ALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHS
                     GSQ"
     primer_bind     complement(5806..5825)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(6232..6341)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        6396..6487
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     6412..6429
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             6505..6528
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             6541..6561
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     misc_recomb     join(7756..7918,1..1195)
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     CDS             complement(join(7772..7918,1..1195))
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="PSLRQSLYNTIAYIERLLNIGTVNDSEITMLIADFYDLYSNYNIE
                     LPPPQALPRSRRPSVVQPAAPAPVPTIVREQTKPEQIIPAAPPPPPSPVPNIPAPPPPP
                     PPSMSELPPAPPMPTEPQPAAPLDDRQQLLEAIRNEKNRTRLRPVKPKTAPETSTIVEV
                     PTVLPKETFEPKPPSASPPPPPPPPPPPAPPAPPPMVDLSSAPPPPPLVDLPSEMLPPP
                     APSLSNVLSELKSGTVRLKPAQKRPQSEIIPKSSTTNLIADVLADTINRRRVAMAKSSS
                     EATSNDEGWDDDDNRPNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVE
                     PLLQNKTQTNIDKARLLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDL
                     IFKFRYKDAENHLIFALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
ORIGIN
        1 atcctcaata gatttgtaaa ataggtttcg attagtttca aacaagggtt gtttttccga
       61 accgatggct ggactatcta atggattttc gctcaacgcc acaaaacttg ccaaatcttg
      121 tagcagcaat ctagctttgt cgatattcgt ttgtgttttg ttttgtaata aaggttcgac
      181 gtcgttcaaa atattatgcg cttttgtatt tctttcatca ctgtcgttag tgtacaattg
      241 actcgacgta aacacgttaa ataaagcttg gacatattta acatcgggcg tgttagcttt
      301 attaggccga ttatcgtcgt cgtcccaacc ctcgtcgtta gaagttgctt ccgaagacga
      361 ttttgccata gccacacgac gcctattaat tgtgtcggct aacacgtccg cgatcaaatt
      421 tgtagttgag ctttttggaa ttatttctga ttgcgggcgt ttttgggcgg gtttcaatct
      481 aactgtgccc gattttaatt cagacaacac gttagaaagc gatggtgcag gcggtggtaa
      541 catttcagac ggcaaatcta ctaatggcgg cggtggtgga gctgatgata aatctaccat
      601 cggtggaggc gcaggcgggg ctggcggcgg aggcggaggc ggaggtggtg gcggtgatgc
      661 agacggcggt ttaggctcaa atgtctcttt aggcaacaca gtcggcacct caactattgt
      721 actggtttcg ggcgccgttt ttggtttgac cggtctgaga cgagtgcgat ttttttcgtt
      781 tctaatagct tccaacaatt gttgtctgtc gtctaaaggt gcagcgggtt gaggttccgt
      841 cggcattggt ggagcgggcg gcaattcaga catcgatggt ggtggtggtg gtggaggcgc
      901 tggaatgtta ggcacgggag aaggtggtgg cggcggtgcc gccggtataa tttgttctgg
      961 tttagtttgt tcgcgcacga ttgtgggcac cggcgcaggc gccgctggct gcacaacgga
     1021 aggtcgtctg cttcgaggca gcgcttgggg tggtggcaat tcaatattat aattggaata
     1081 caaatcgtaa aaatctgcta taagcattgt aatttcgcta tcgtttaccg tgccgatatt
     1141 taacaaccgc tcaatgtaag caattgtatt gtaaagagat tgtctcaagc tcggatcgat
     1201 cccgcacgcc gataacaagc cttttcattt ttactacagc attgtagtgg cgagacactt
     1261 cgctgtcgtc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     1321 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1381 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1441 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1501 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     1561 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1621 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1681 cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1741 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     1801 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     1861 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     1921 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     1981 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2041 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2101 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2161 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     2221 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     2281 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     2341 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     2401 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     2461 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     2521 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     2581 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     2641 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     2701 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     2761 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     2821 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     2881 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     2941 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3001 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3061 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3121 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3181 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3241 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3301 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3361 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3421 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3481 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3541 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3601 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3661 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3721 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3781 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3841 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3901 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3961 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     4021 ggccgattca ttaatgcaga acggctccgc ccactattaa tgaaattaaa aattccaatt
     4081 ttaaaaaacg cagcaagaga aacatttgta tgaaagaatg cgtagaagga aagaaaaatg
     4141 tcgtcgacat gctgaacaac aagattaata tgcctccgtg tataaaaaaa atattgaacg
     4201 atttgaaaga aaacaatgta ccgcgcggcg gtatgtacag gaagaggttt atactaaact
     4261 gttacattgc aaacgtggtt tcgtgtgcca agtgtgaaaa ccgatgttta atcaaggctc
     4321 tgacgcattt ctacaaccac gactccaagt gtgtgggtga agtcatgcat cttttaatca
     4381 aatcccaaga tgtgtataaa ccaccaaact gccaaaaaat gaaaactgtc gacaagctct
     4441 gtccgtttgc tggcaactgc aagggtctca atcctatttg taattattga ataataaaac
     4501 aattataaat gtcaaatttg ttttttatta acgatacaaa ccaaacgcaa caagaacatt
     4561 tgtagtatta tctataattg aaaacgcgta gttataatcg ctgaggtaat atttaaaatc
     4621 attttcaaat gattcacagt taatttgcga caatataatt ttattttcac ataaactaga
     4681 cgccttgtcg tcttcttctt cgtattcctt ctctttttca tttttctcct cataaaaatt
     4741 aacatagtta ttatcgtatc catatatgta tctatcgtat agagtaaatt ttttgttgtc
     4801 ataaatatat atgtcttttt taatggggtg tatagtaccg ctgcgcatag tttttctgta
     4861 atttacaaca gtgctatttt ctggtagttc ttcggagtgt gttgctttaa ttattaaatt
     4921 tatataatca atgaatttgg gatcgtcggt tttgtacaat atgttgccgg catagtacgc
     4981 agcttcttct agttcaatta caccattttt tagcagcacc ggattaacat aactttccaa
     5041 aatgttgtac gaaccgttaa acaaaaacag ttcacctccc ttttctatac tattgtctgc
     5101 gagcagttgt ttgttgttaa aaataacagc cattgtaatg agacgcacaa actaatatca
     5161 caaactggaa atgtctatca atatatagtt gctgatataa cttcgtatag catacattat
     5221 acgaagttat ctggttgaaa cgtacccgta gtggctatgg cagggcttgc cgccccgacg
     5281 ttggctgcga gccctgggcc ttcacccgaa cttgggggtt ggggtgggga aaaggaagaa
     5341 acgcgggcgt attggtccca atggggtctc ggtggggtat cgacagagtg ccagccctgg
     5401 gaccgaaccc cgcgtttatg aacaaacgac ccaacacccg tgcgttttat tctgtctttt
     5461 tattgccgtc atagcgcggg ttccttccgg tattgtctcc ttccgtgttt cagttagcct
     5521 cccccatctc ccgctattac tcgagtcact gggagccgga gtggcgggcc tcggcgtgct
     5581 cgtactgctc caccacggtg tagtcctcgt tgtggttggt gatgtccagc ttggagtcca
     5641 cgtagtggtt gccgggcagc tgcacgggct tcttggcctt gtacacggtc ttgaagtcgc
     5701 aggtgtagtg gccgccgtcc ttcagcttca gggcgtggga gatctcgccc ttcagcacgc
     5761 cgtcctgggg gtacagcttc tcggtggagg gctcccagcc ggcagtcttc ttctgcatta
     5821 cggggccgtc ggcggggaag ttcacgccct tgaacttcac cttgtagatg aaggtgccgt
     5881 cctgcaggga ggagtcctgc tgcacctcca ccacgccgcc gtcctcgaag ttcatggagc
     5941 gctcccaggt gaagccctcg gggaaggaca gcttcatgta gtcggggatg tcggcggggt
     6001 gcttcacgta ggccttggag ccgtactgga actgggggga caggatgtcc caggcgaagg
     6061 gcagggggcc gcccttggtc acctgcagct tggcggtctg ggtgccctcg tagggcttgc
     6121 cctcgccctc gccctcgatc tcgaagtagt ggccgttcac ggagccctcc atgcgcacct
     6181 tgaactgcat gaactccttg atgacgtcct cggtgttgtc catggtagag tgattgtaaa
     6241 taaaatgtaa tttacagtat agtattttaa ttaatataca aatgatttga taataattct
     6301 tatttaacta taatatattg tgttgggttg aattaaaggt ccgtatacta gtatcgattc
     6361 gcgacctact ccggaatatt aatagagttg ctgatatcat ggagataatt aaaatgataa
     6421 ccatctcgca aataaataag tattttactg ttttcgtaac agttttgtaa taaaaaaacc
     6481 tataaatacg gatctaccat ggactggagc cacccgcagt tcgaaaaatc cgcggtcgac
     6541 gagaacctct acttccaggg aggcggccgc gactttgatg tcaagcgcaa atatttccgc
     6601 caagagctgg agcgtttaga tgaggggctc cggaaagaag acatggctgt gcatgtccgt
     6661 cgtgaccatg tgtttgaaga ctcctatcgt gagctgcatc gcaaatcccc cgaagaaatg
     6721 aagaatcgat tgtatatagt atttgaagga gaagaagggc aggatgctgg cgggctcctg
     6781 cgggagtggt atatgatcat ctctcgagag atgtttaacc ctatgtatgc cttgttccgt
     6841 acctcacctg gtgatcgagt cacctacacc atcaatccat cttcccactg caaccccaac
     6901 catctgtcct attttaaatt tgtcggacgc attgtggcca aagctgtata tgacaaccgt
     6961 cttctggagt gctactttac tcgatccttt tacaaacaca tcttgggcaa gtcagtcaga
     7021 tatacagata tggagagtga agattaccac ttctaccaag gtctggttta tctgctggaa
     7081 aatgatgtct ccacactagg ctatgacctc accttcagca ctgaggtcca agagtttgga
     7141 gtttgtgaag ttcgtgacct caaacccaat ggggccaaca tcttggtaac agaggagaat
     7201 aagaaggagt atgtacacct ggtatgccag atgagaatga caggagccat ccgcaagcag
     7261 ttggcggctt tcttagaagg cttctatgag atcattccaa agcgcctcat ttccatcttc
     7321 actgagcagg agttagagct gcttatatca ggactgccca ccattgacat cgatgatctg
     7381 aaatccaaca ctgaatacca caagtaccag tccaactcta ttcagatcca gtggttctgg
     7441 agagcattgc gttctttcga tcaagctgac cgtgccaagt tcctccagtt tgtcacgggt
     7501 acttccaagg tacccctgca aggctttgct gccctcgaag gcatgaatgg cattcagaag
     7561 tttcagatcc atcgagatga caggtccaca gatcgcctgc cttcagctca cacatgtttt
     7621 aatcagctgg atctgcctgc ctatgagagc tttgagaagc tccgccacat gctactgttg
     7681 gctatccagg agtgctctga aggctttggg ctggcctaag gtacccccgg acggccgctt
     7741 aattaattga tccgggttat tagtacattt attaagcgct agattctgtg cgttgttgat
     7801 ttacagacaa ttgttgtacg tattttaata attcattaaa tttataatct ttagggtggt
     7861 atgttagagc gaaaatcaaa tgattttcag cgtctttata tctgaattta aatattaa
//
