LOCUS       sequence-372353-        6857 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express GST-tagged NEDD4L W2-W3-W4-HECT (348) in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6857)
  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 6857)
  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: pGEX6P1-NEDD4L_W2-W3-W4-HECT (348)
FEATURES             Location/Qualifiers
     source          1..6857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(101..106,107..122,123..129)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    137..153
                     /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)."
     CDS             176..829
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     787..809
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             836..859
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2825..2847)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2992..3010)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3078..3182
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3183..3251,3252..4043)
                     /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(3401..3420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4214..4802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4703..4722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4956..4973
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5046..5123
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5124..6206
                     /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(5143..5162)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6255..6260,6261..6278,6279..6285)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6293..6309
                     /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     6317..6333
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6317..6333
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6329..6856
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6349..6366)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6349..6365)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6358..6380)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tcactgcata attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc
       61 gacatcataa cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct
      121 cgtataatgt gtggaattgt gagcggataa caatttcaca caggaaacag tattcatgtc
      181 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      241 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
      301 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      361 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
      421 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
      481 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
      541 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
      601 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
      661 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
      721 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
      781 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctgga
      841 agttctgttc caggggcccc tgggagtacg cgccggctcc gaacagggcc atctaccacc
      901 gcccagtgcc ccagctggga gagcgcgttc atcaactgtc acgggtggtg aggaaccaac
      961 gccatcagtg gcctatgtac atactacgcc gggtctgcct tcaggctggg aagaaagaaa
     1021 agatgctaag gggcgcacat actatgtcaa tcataacaat cgaaccacaa cttggactcg
     1081 acctatcatg cagcttgcag aagatggtgc gtccggatca gccacaaaca gtaacaacca
     1141 tctaatcgag cctcagatcc gccggcctcg tagcctcagc tcgccaacag taactttatc
     1201 tgccccgctg gagggtgcca aggactcacc cgtacgtcgg gctgtgaaag acaccctttc
     1261 caacccacag tccccacagc catcacctta caactccccc aaaccacaac acaaagtcac
     1321 acagagcttc ttgccacccg gctgggaaat gaggatagcg ccaaacggcc ggcccttctt
     1381 cattgatcat aacacaaaga ctacaacctg ggaagatcca cgtttgaaat ttccagtaca
     1441 tatgcggtca aagacatctt taaaccccaa tgaccttggc ccccttcctc ctggctggga
     1501 agaaagaatt cacttggatg gccgaacgtt ttatattgat cataatagca aaattactca
     1561 gtgggaagac ccaagactgc agaacccagc tattactggt ccggctgtcc cttactccag
     1621 agaatttaag cagaaatatg actacttcag gaagaaatta aagaaacctg ctgatatccc
     1681 caataggttt gaaatgaaac ttcacagaaa taacatattt gaagagtcct atcggagaat
     1741 tatgtccgtg aaaagaccag atgtcctaaa agctagactg tggattgagt ttgaatcaga
     1801 gaaaggtctt gactatgggg gtgtggccag agaatggttc ttcttactgt ccaaagagat
     1861 gttcaacccc tactacggcc tctttgagta ctctgccacg gacaactaca cccttcagat
     1921 caaccctaat tcaggcctct gtaatgagga tcatttgtcc tacttcactt ttattggaag
     1981 agttgctggt ctggccgtat ttcatgggaa gctcttagat ggtttcttca ttagaccatt
     2041 ttacaagatg atgttgggaa agcagataac cctgaatgac atggaatctg tggatagtga
     2101 atattacaac tctttgaaat ggatcctgga gaatgaccct actgagctgg acctcatgtt
     2161 ctgcatagac gaagaaaact ttggacagac atatcaagtg gatttgaagc ccaatgggtc
     2221 agaaataatg gtcacaaatg aaaacaaaag ggaatatatc gacttagtca tccagtggag
     2281 atttgtgaac agggtccaga agcagatgaa cgccttcttg gagggattca cagaactact
     2341 tcctattgat ttgattaaaa tttttgatga aaatgagctg gagttgctca tgtgcggcct
     2401 cggtgatgtg gatgtgaatg actggagaca gcattctatt tacaagaacg gctactgccc
     2461 aaaccacccc gtcattcagt ggttctggaa ggctgtgcta ctcatggacg ccgaaaagcg
     2521 tatccggtta ctgcagtttg tcacagggac atcgcgagta cctatgaatg gatttgccga
     2581 actttatggt tccaatggtc ctcagctatt tacaatagag caatggggca gtcctgagaa
     2641 actgcccaga gctcacacat gctttaatcg ccttgactta cctccatatg aaacctttga
     2701 agatttacga gagaaacttc tcatggccgt ggaaaatgct caaggatttg aaggggtgga
     2761 ttaagcggcc gcatcgtgac tgactgacga tctgcctcgc gcgtttcggt gatgacggtg
     2821 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     2881 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     2941 tgacccagtc acgtagcgat agcggagtgt ataattcttg aagacgaaag ggcctcgtga
     3001 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     3061 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3121 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3181 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3241 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3301 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3361 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3421 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3481 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3541 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3601 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3661 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3721 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3781 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     3841 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     3901 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     3961 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4021 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4081 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4141 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4201 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4261 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4321 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4381 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4441 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4501 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4561 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4621 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4681 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4741 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4801 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4861 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4921 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4981 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcataa
     5041 attccgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     5101 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     5161 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     5221 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     5281 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     5341 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     5401 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     5461 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     5521 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     5581 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     5641 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     5701 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     5761 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     5821 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     5881 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     5941 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     6001 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     6061 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacg ctagcgccca
     6121 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     6181 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     6241 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
     6301 ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac tggccgtcgt
     6361 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca
     6421 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     6481 gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt ccggcaccag aagcggtgcc
     6541 ggaaagctgg ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc cctcaaactg
     6601 gcagatgcac ggttacgatg cgcccatcta caccaacgtg acctatccca ttacggtcaa
     6661 tccgccgttt gttcccacgg agaatccgac gggttgttac tcgctcacat ttaatgttga
     6721 tgaaagctgg ctacaggaag gccagacgcg aattattttt gatggcgttg gaattagctt
     6781 atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag ctgtggtatg
     6841 gctgtgcagg tcgtaaa
//
