LOCUS       sequence-372459-        7077 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express his-tagged NEDD4L W2-W3-W4-HECT in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7077)
  AUTHORS   Wright KM, Nathan S, Jiang H, Xia W, Kim H, Chakouri N, Nwafor JN, 
            Fossier L, Srinivasan L, Chen Z, Boronina T, Post J, Paul S, Cole 
            RN, Ben-Johny M, Cole PA, Gabelli SB
  TITLE     NEDD4L intramolecular interactions regulate its auto and substrate 
            Na(V)1.5 ubiquitination.
  JOURNAL   J Biol Chem. 2024 Mar;300(3):105715. doi: 10.1016/j.jbc.2024.105715.
            Epub 2024 Feb 2.
  PUBMED    38309503
REFERENCE   2  (bases 1 to 7077)
  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: "J Biol 
            Chem."; date: "2024-03"; volume: "300(3):105715. doi"; pages: " 
            10.1016/j.jbc.2024.105715. Epub 2024 Feb 2"
COMMENT     Sequence Label: pET28a(+)-NEDD4L_W2-W3-W4-HECT (381)
FEATURES             Location/Qualifiers
     source          1..7077
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1961..1984)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(1988..2005)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2018..2040
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2055..2079
                     /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(2079..2098)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2080..2098)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2182..2201)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2407..2484
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2485..3567
                     /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     complement(2504..2523)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4376..4567
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4561..4583)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4669..4808
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4724..4743
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4823..4840)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4994..5582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5074..5093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5693..5712)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5704..6519
                     /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             5704..6519
                     /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     complement(5777..5796)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6611..7066)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6748..6769)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6960..6979
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcttaatcca
      181 ccccttcaaa tccttgagca ttttccacgg ccatgagaag tttctctcgt aaatcttcaa
      241 aggtttcata tggaggtaag tcaaggcgat taaagcatgt gtgagctctg ggcagtttct
      301 caggactgcc ccattgctct attgtaaata gctgaggacc attggaacca taaagttcgg
      361 caaatccatt cataggtact cgcgatgtcc ctgtgacaaa ctgcagtaac cggatacgct
      421 tttcggcgtc catgagtagc acagccttcc agaaccactg aatgacgggg tggtttgggc
      481 agtagccgtt cttgtaaata gaatgctgtc tccagtcatt cacatccaca tcaccgaggc
      541 cgcacatgag caactccagc tcattttcat caaaaatttt aatcaaatca ataggaagta
      601 gttctgtgaa tccctccaag aaggcgttca tctgcttctg gaccctgttc acaaatctcc
      661 actggatgac taagtcgata tattcccttt tgttttcatt tgtgaccatt atttctgacc
      721 cattgggctt caaatccact tgatatgtct gtccaaagtt ttcttcgtct atgcagaaca
      781 tgaggtccag ctcagtaggg tcattctcca ggatccattt caaagagttg taatattcac
      841 tatccacaga ttccatgtca ttcagggtta tctgctttcc caacatcatc ttgtaaaatg
      901 gtctaatgaa gaaaccatct aagagcttcc catgaaatac ggccagacca gcaactcttc
      961 caataaaagt gaagtaggac aaatgatcct cattacagag gcctgaatta gggttgatct
     1021 gaagggtgta gttgtccgtg gcagagtact caaagaggcc gtagtagggg ttgaacatct
     1081 ctttggacag taagaagaac cattctctgg ccacaccccc atagtcaaga cctttctctg
     1141 attcaaactc aatccacagt ctagctttta ggacatctgg tcttttcacg gacataattc
     1201 tccgatagga ctcttcaaat atgttatttc tgtgaagttt catttcaaac ctattgggga
     1261 tatcagcagg tttctttaat ttcttcctga agtagtcata tttctgctta aattctctgg
     1321 agtaagggac agccggacca gtaatagctg ggttctgcag tcttgggtct tcccactgag
     1381 taattttgct attatgatca atataaaacg ttcggccatc caagtgaatt ctttcttccc
     1441 agccaggagg aagggggcca aggtcattgg ggtttaaaga tgtctttgac cgcatatgta
     1501 ctggaaattt caaacgtgga tcttcccagg ttgtagtctt tgtgttatga tcaatgaaga
     1561 agggccggcc gtttggcgct atcctcattt cccagccggg tggcaagaag ctctgtgtga
     1621 ctttgtgttg tggtttgggg gagttgtaag gtgatggctg tggggactgt gggttggaaa
     1681 gggtgtcttt cacagcccga cgtacgggtg agtccttggc accctccagc ggggcagata
     1741 aagttactgt tggcgagctg aggctacgag gccggcggat ctgaggctcg attagatggt
     1801 tgttactgtt tgtggctgat ccggacgcac catcttctgc aagctgcatg ataggtcgag
     1861 tccaagttgt ggttcgattg ttatgattga catagtatgt gcgcccctta gcatcttttc
     1921 tttcttccca gcctgaaggc agacccggcg tggtatgtac tgggccctgg aacagcacct
     1981 ccaggccgtg gtggtggtgg tggtgcccca tggtatatct ccttcttaaa gttaaacaaa
     2041 attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat
     2101 ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg
     2161 ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc
     2221 gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg
     2281 ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg
     2341 gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag
     2401 atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     2461 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     2521 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     2581 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     2641 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     2701 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     2761 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     2821 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     2881 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     2941 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     3001 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     3061 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     3121 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     3181 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     3241 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     3301 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     3361 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     3421 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     3481 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     3541 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     3601 ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg
     3661 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact
     3721 cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag
     3781 cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc
     3841 cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat
     3901 ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg
     3961 gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg
     4021 ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc
     4081 gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
     4141 aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg
     4201 accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca
     4261 acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg
     4321 tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac
     4381 caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct
     4441 tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca
     4501 cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     4561 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     4621 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     4681 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     4741 gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     4801 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4861 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4921 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4981 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5041 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5101 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5161 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     5221 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     5281 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     5341 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5401 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     5461 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5521 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5581 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5641 aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa tacaaggggt
     5701 gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg
     5761 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     5821 atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     5881 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     5941 cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt actcaccact
     6001 gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc aggtgaaaat
     6061 attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt
     6121 ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat gaataacggt
     6181 ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga acaagtctgg
     6241 aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca tggtgatttc
     6301 tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga tgttggacga
     6361 gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct cggtgagttt
     6421 tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc tgatatgaat
     6481 aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca tgagcggata
     6541 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6601 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     6661 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     6721 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     6781 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     6841 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     6901 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     6961 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     7021 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgcca
//
