LOCUS       sequence-372454-        7664 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express GST-tagged NEDD4L FL with removal of the linker between the 
            WW2 and WW3 domains in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7664)
  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 7664)
  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_Delta-2,3-linker
FEATURES             Location/Qualifiers
     source          1..7664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(117..122,123..138,139..145)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    153..169
                     /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             192..845
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     803..825
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             852..875
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3648..3670)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3815..3833)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3901..4005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4006..4074,4075..4866)
                     /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(4224..4243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5037..5625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5526..5545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5779..5796
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5869..5946
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5947..7029
                     /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(5966..5985)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7078..7083,7084..7101,7102..7108)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7116..7132
                     /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     7140..7156
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7140..7156
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(7152..7664,1..15)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(7172..7189)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7172..7188)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7181..7203)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga
       61 taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat gagctgttga
      121 caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg
      181 aaacagtatt catgtcccct atactaggtt attggaaaat taagggcctt gtgcaaccca
      241 ctcgacttct tttggaatat cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg
      301 aaggtgataa atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttcctt
      361 attatattga tggtgatgtt aaattaacac agtctatggc catcatacgt tatatagctg
      421 acaagcacaa catgttgggt ggttgtccaa aagagcgtgc agagatttca atgcttgaag
      481 gagcggtttt ggatattaga tacggtgttt cgagaattgc atatagtaaa gactttgaaa
      541 ctctcaaagt tgattttctt agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt
      601 tatgtcataa aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatg
      661 acgctcttga tgttgtttta tacatggacc caatgtgcct ggatgcgttc ccaaaattag
      721 tttgttttaa aaaacgtatt gaagctatcc cacaaattga taagtacttg aaatccagca
      781 agtatatagc atggcctttg cagggctggc aagccacgtt tggtggtggc gaccatcctc
      841 caaaatcgga tctggaagtt ctgttccagg ggcccgtacg cgccggctcc atggcgaccg
      901 ggctcgggga gccggtctat ggactttccg aagacgaggg agagtcccgt attctcagag
      961 taaaagttgt ttctggaatt gatctcgcca aaaaggacat ctttggagcc agtgatccgt
     1021 atgtgaaact ttcattgtac gtagcggatg agaatagaga acttgctttg gtccagacaa
     1081 aaacaattaa aaagacactg aacccaaaat ggaatgaaga attttatttc agggtaaacc
     1141 catctaatca cagactccta tttgaagtat ttgacgaaaa tagactgaca cgagacgact
     1201 tcctgggcca ggtggacgtg ccccttagtc accttccgac agaagatcca accatggagc
     1261 gaccctatac atttaaggac tttctcctca gaccaagaag tcataagtct cgagttaagg
     1321 gatttttgcg attgaaaatg gcctatatgc caaaaaatgg aggtcaagat gaagaaaaca
     1381 gtgaccagag ggatgacatg gagcatggat gggaagttgt tgactcaaat gactcggctt
     1441 ctcagcacca agaggaactt cctcctcctc ctctgcctcc cgggtgggaa gaaaaagtgg
     1501 acaatttagg ccgaacttac tatgtcaacc acaacaaccg gaccactcag tggcacagac
     1561 caagcctgat ggacgtgtcc tcggagtcgg acaataacat cagacagatc aaccaggagg
     1621 cagcacaccg gcgcttccgc tcccgcaggc acatcagcga agacttggag cccgagccct
     1681 cggagggcgg ggatgtcccc gagccttggg agaccatttc agaggaagtg aatatcgctg
     1741 gagactctct cggtctggct ctgcccccac caccggcctc cccaggatct cggaccagcc
     1801 ctcaggagct gtcagaggaa ctaagcagaa ggcttcagat cactccagac tccaatgggg
     1861 aacagttcag ctctttgatt caaagagaac cctcctcaag gttgaggtca tgcagtgtca
     1921 ccgacgcagt tgcagaacag ggccatctac caccgcccag tgccccagct gggagagcgc
     1981 gttcatcaac tgtcacgggt ggtgaggaac caacgccatc agtggcctat gtacatacta
     2041 cgccgggtct gccttcaggc tgggaagaaa gaaaagatgc taaggggcgc acatactatg
     2101 tcaatcataa caatcgaacc acaacttgga ctcgacctat catgcagagc ttcttgccac
     2161 ccggctggga aatgaggata gcgccaaacg gccggccctt cttcattgat cataacacaa
     2221 agactacaac ctgggaagat ccacgtttga aatttccagt acatatgcgg tcaaagacat
     2281 ctttaaaccc caatgacctt ggcccccttc ctcctggctg ggaagaaaga attcacttgg
     2341 atggccgaac gttttatatt gatcataata gcaaaattac tcagtgggaa gacccaagac
     2401 tgcagaaccc agctattact ggtccggctg tcccttactc cagagaattt aagcagaaat
     2461 atgactactt caggaagaaa ttaaagaaac ctgctgatat ccccaatagg tttgaaatga
     2521 aacttcacag aaataacata tttgaagagt cctatcggag aattatgtcc gtgaaaagac
     2581 cagatgtcct aaaagctaga ctgtggattg agtttgaatc agagaaaggt cttgactatg
     2641 ggggtgtggc cagagaatgg ttcttcttac tgtccaaaga gatgttcaac ccctactacg
     2701 gcctctttga gtactctgcc acggacaact acacccttca gatcaaccct aattcaggcc
     2761 tctgtaatga ggatcatttg tcctacttca cttttattgg aagagttgct ggtctggccg
     2821 tatttcatgg gaagctctta gatggtttct tcattagacc attttacaag atgatgttgg
     2881 gaaagcagat aaccctgaat gacatggaat ctgtggatag tgaatattac aactctttga
     2941 aatggatcct ggagaatgac cctactgagc tggacctcat gttctgcata gacgaagaaa
     3001 actttggaca gacatatcaa gtggatttga agcccaatgg gtcagaaata atggtcacaa
     3061 atgaaaacaa aagggaatat atcgacttag tcatccagtg gagatttgtg aacagggtcc
     3121 agaagcagat gaacgccttc ttggagggat tcacagaact acttcctatt gatttgatta
     3181 aaatttttga tgaaaatgag ctggagttgc tcatgtgcgg cctcggtgat gtggatgtga
     3241 atgactggag acagcattct atttacaaga acggctactg cccaaaccac cccgtcattc
     3301 agtggttctg gaaggctgtg ctactcatgg acgccgaaaa gcgtatccgg ttactgcagt
     3361 ttgtcacagg gacatcgcga gtacctatga atggatttgc cgaactttat ggttccaatg
     3421 gtcctcagct atttacaata gagcaatggg gcagtcctga gaaactgccc agagctcaca
     3481 catgctttaa tcgccttgac ttacctccat atgaaacctt tgaagattta cgagagaaac
     3541 ttctcatggc cgtggaaaat gctcaaggat ttgaaggggt ggattaagcg gccgcatcgt
     3601 gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3661 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3721 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     3781 gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     3841 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3901 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3961 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4021 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     4081 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     4141 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     4201 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     4261 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     4321 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     4381 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     4441 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     4501 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     4561 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     4621 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     4681 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     4741 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4801 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4861 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4921 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4981 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     5041 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     5101 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     5161 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     5221 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     5281 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5341 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5401 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5461 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5521 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5581 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5641 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5701 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     5761 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     5821 tattttctcc ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa
     5881 tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg
     5941 gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag
     6001 accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg
     6061 gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc
     6121 aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa
     6181 attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg
     6241 gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc
     6301 gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct
     6361 gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt
     6421 attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt
     6481 caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg
     6541 gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc
     6601 gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt
     6661 cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc
     6721 gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac
     6781 agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa
     6841 accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg
     6901 ttgcccgtct cactggtgaa aagaaaaacc acgctagcgc ccaatacgca aaccgcctct
     6961 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     7021 gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt
     7081 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     7141 aggaaacagc tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact
     7201 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     7261 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     7321 gcgaatggcg ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt
     7381 gcgatcttcc tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg
     7441 atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca
     7501 cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg
     7561 aaggccagac gcgaattatt tttgatggcg ttggaattag cttatcgact gcacggtgca
     7621 ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt atgg
//
