LOCUS       sequence-372422-        7901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express GST-tagged NEDD4L FL in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7901)
  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 7901)
  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_FL
FEATURES             Location/Qualifiers
     source          1..7901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(111..116,117..132,133..139)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    147..163
                     /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             186..839
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     797..819
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             846..869
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(3879..3901)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4046..4064)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4132..4236
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4237..4305,4306..5097)
                     /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(4455..4474)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5268..5856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5757..5776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6010..6027
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        6100..6177
                     /gene="lacI"
                     /label=lacI promoter
     CDS             6178..7260
                     /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(6197..6216)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(7309..7314,7315..7332,7333..7339)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7347..7363
                     /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     7371..7387
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7371..7387
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(7383..7901,1..9)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(7403..7420)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7403..7419)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7412..7434)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc tggataatgt
       61 tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg ttgacaatta
      121 atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca caggaaacag
      181 tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac
      241 ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg
      301 ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata
      361 ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc
      421 acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg
      481 ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca
      541 aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc
      601 ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc
      661 ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt
      721 ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata
      781 tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat
      841 cggatctgga agttctgttc caggggcccc tgggagtacg 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 catgcagctt gcagaagatg
     2161 gtgcgtccgg atcagccaca aacagtaaca accatctaat cgagcctcag atccgccggc
     2221 ctcgtagcct cagctcgcca acagtaactt tatctgcccc gctggagggt gccaaggact
     2281 cacccgtacg tcgggctgtg aaagacaccc tttccaaccc acagtcccca cagccatcac
     2341 cttacaactc ccccaaacca caacacaaag tcacacagag cttcttgcca cccggctggg
     2401 aaatgaggat agcgccaaac ggccggccct tcttcattga tcataacaca aagactacaa
     2461 cctgggaaga tccacgtttg aaatttccag tacatatgcg gtcaaagaca tctttaaacc
     2521 ccaatgacct tggccccctt cctcctggct gggaagaaag aattcacttg gatggccgaa
     2581 cgttttatat tgatcataat agcaaaatta ctcagtggga agacccaaga ctgcagaacc
     2641 cagctattac tggtccggct gtcccttact ccagagaatt taagcagaaa tatgactact
     2701 tcaggaagaa attaaagaaa cctgctgata tccccaatag gtttgaaatg aaacttcaca
     2761 gaaataacat atttgaagag tcctatcgga gaattatgtc cgtgaaaaga ccagatgtcc
     2821 taaaagctag actgtggatt gagtttgaat cagagaaagg tcttgactat gggggtgtgg
     2881 ccagagaatg gttcttctta ctgtccaaag agatgttcaa cccctactac ggcctctttg
     2941 agtactctgc cacggacaac tacacccttc agatcaaccc taattcaggc ctctgtaatg
     3001 aggatcattt gtcctacttc acttttattg gaagagttgc tggtctggcc gtatttcatg
     3061 ggaagctctt agatggtttc ttcattagac cattttacaa gatgatgttg ggaaagcaga
     3121 taaccctgaa tgacatggaa tctgtggata gtgaatatta caactctttg aaatggatcc
     3181 tggagaatga ccctactgag ctggacctca tgttctgcat agacgaagaa aactttggac
     3241 agacatatca agtggatttg aagcccaatg ggtcagaaat aatggtcaca aatgaaaaca
     3301 aaagggaata tatcgactta gtcatccagt ggagatttgt gaacagggtc cagaagcaga
     3361 tgaacgcctt cttggaggga ttcacagaac tacttcctat tgatttgatt aaaatttttg
     3421 atgaaaatga gctggagttg ctcatgtgcg gcctcggtga tgtggatgtg aatgactgga
     3481 gacagcattc tatttacaag aacggctact gcccaaacca ccccgtcatt cagtggttct
     3541 ggaaggctgt gctactcatg gacgccgaaa agcgtatccg gttactgcag tttgtcacag
     3601 ggacatcgcg agtacctatg aatggatttg ccgaacttta tggttccaat ggtcctcagc
     3661 tatttacaat agagcaatgg ggcagtcctg agaaactgcc cagagctcac acatgcttta
     3721 atcgccttga cttacctcca tatgaaacct ttgaagattt acgagagaaa cttctcatgg
     3781 ccgtggaaaa tgctcaagga tttgaagggg tggattaagc ggccgcatcg tgactgactg
     3841 acgatctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3901 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3961 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     4021 gtgtataatt cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
     4081 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     4141 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4201 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     4261 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     4321 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     4381 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     4441 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
     4501 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     4561 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     4621 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     4681 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     4741 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     4801 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4861 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4921 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4981 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     5041 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     5101 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5161 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5221 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5281 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5341 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5401 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5461 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5521 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5581 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5641 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5701 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5761 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5821 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5881 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5941 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6001 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     6061 cttacgcatc tgtgcggtat ttcacaccgc ataaattccg acaccatcga atggcgcaaa
     6121 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     6181 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     6241 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     6301 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     6361 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     6421 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     6481 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     6541 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     6601 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     6661 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     6721 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     6781 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     6841 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     6901 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     6961 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     7021 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     7081 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     7141 tcactggtga aaagaaaaac cacgctagcg cccaatacgc aaaccgcctc tccccgcgcg
     7201 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     7261 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccccaggctt tacactttat
     7321 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     7381 ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     7441 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     7501 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     7561 gctttgcctg gtttccggca ccagaagcgg tgccggaaag ctggctggag tgcgatcttc
     7621 ctgaggccga tactgtcgtc gtcccctcaa actggcagat gcacggttac gatgcgccca
     7681 tctacaccaa cgtgacctat cccattacgg tcaatccgcc gtttgttccc acggagaatc
     7741 cgacgggttg ttactcgctc acatttaatg ttgatgaaag ctggctacag gaaggccaga
     7801 cgcgaattat ttttgatggc gttggaatta gcttatcgac tgcacggtgc accaatgctt
     7861 ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg c
//
